Method for detecting components of irinotecan hydrochloride injection based on liquid chromatography

Abstract

The invention belongs to the technical field of drug analysis and detection, and particularly relates to a liquid chromatography-based irinotecan hydrochloride injection component detection method, which comprises the following steps: S100, sample pretreatment: taking an irinotecan hydrochloride injection, diluting with a 0.1% formic acid aqueous solution, and filtering with a 0.22 [mu] m hydrophilic PTFE filter membrane to obtain a test solution; step S200, chromatographic detection: carrying out gradient elution by adopting a core-shell C18 chromatographic column, taking a 0.05% trifluoroacetic acid aqueous solution as a mobile phase A and taking acetonitrile as a mobile phase B; step S300, performing dual-wavelength detection: performing synchronous detection under the wavelengths of 254 nm and 280 nm, and recording a chromatogram; step S400, quantitative analysis: calculating the content of each component through an external standard method, combining a core-shell chromatographic column with a gradient elution procedure to realize complete separation of main components irinotecan hydrochloride, process impurities, degradation impurities and SN-38, and determining the content of irinotecan hydrochloride by optimizing chromatographic conditions, a mobile phase system and a pretreatment process. And comprehensive, rapid and high-sensitivity separation and detection of main components, process impurities, degradation impurities and related substances are realized.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis and detection, and specifically relates to a method for detecting components of irinotecan hydrochloride injection based on liquid chromatography. Background Art

[0002] Irinotecan hydrochloride is a topoisomerase I inhibitor widely used in chemotherapy for solid tumors such as advanced colorectal cancer and ovarian cancer. The quality control of its injection is directly related to the safety and efficacy of clinical medication, so strict testing of impurities is required. According to the "Chinese Pharmacopoeia" (2020 edition) and related guidelines, the quality standards of irinotecan hydrochloride injection must control the content of the main components, related substances (including process impurities, degradation impurities) and residual solvents. Currently, the component detection of irinotecan hydrochloride injection mainly relies on high-performance liquid chromatography (HPLC), but the existing technology has the following defects:

[0003] Traditional C18 chromatographic columns have poor separation effects on highly polar impurities (such as irinotecan's hydrolysis product 7-ethyl-10-hydroxycamptothecin, SN-38) and the main component, and peak overlap is prone to occur; conventional ultraviolet detectors (UV) have insufficient response values ​​for low-concentration impurities at a wavelength of 254nm, making it difficult to meet the detection requirements of trace impurities (such as a single impurity ≤0.1%); existing methods mostly target the main component content or single impurities (such as SN-38), and lack comprehensive coverage of process impurities (such as synthetic intermediates and by-products), degradation impurities (such as photolysis and oxidation products) and related substances; conventional direct injection or simple dilution can easily introduce matrix interference (such as buffer salts and surfactants in the injection solution), affecting chromatographic peak shape and detection accuracy.

[0004] Therefore, it is urgent to develop a liquid chromatography-based component detection method for irinotecan hydrochloride injection to meet strict quality control requirements. Summary of the Invention

[0005] To address the problems existing in the above-mentioned background technology, the present invention proposes a method for detecting the components of irinotecan hydrochloride injection based on liquid chromatography. By optimizing the chromatographic conditions, mobile phase system and pretreatment process, comprehensive, rapid and highly sensitive separation and detection of the main components, process impurities, degradation impurities and related substances are achieved, thereby solving the problems of low separation efficiency, insufficient sensitivity and limited multi-component detection capability in the existing technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The method and method for detecting the components of irinotecan hydrochloride injection based on liquid chromatography include the following steps:

[0008] Step S100, sample pretreatment: taking irinotecan hydrochloride injection, diluting it with 0.1% formic acid aqueous solution, and filtering it through a 0.22 μm hydrophilic PTFE filter membrane to obtain a test solution;

[0009] Step S200, chromatographic detection: using a core-shell C18 chromatographic column, with 0.05% trifluoroacetic acid aqueous solution as mobile phase A and acetonitrile as mobile phase B, performing gradient elution;

[0010] Step S300, dual wavelength detection: simultaneous detection at wavelengths of 254 nm and 280 nm, and recording of chromatograms;

[0011] Step S400, quantitative analysis: calculating the content of each component by external standard method.

[0012] Preferably, the specifications of the core-shell C18 chromatographic column are: inner diameter 2.1 mm, column length 100 mm, and filler particle size 1.7 μm.

[0013] Preferably, the gradient elution procedure is:

[0014] 0-2 min, mobile phase B volume fraction 10%;

[0015] From 2 to 8 min, the volume fraction of mobile phase B was linearly increased to 30%;

[0016] From 8 to 15 min, the volume fraction of mobile phase B was linearly increased to 50%;

[0017] From 15 to 18 min, the volume fraction of mobile phase B increased linearly to 90%;

[0018] 18-20 min, the volume fraction of mobile phase B was maintained at 90%;

[0019] From 20 to 22 min, the volume fraction of mobile phase B decreased linearly to 10%.

[0020] Preferably, the column flow rate is 0.35 mL / min and the column temperature is 35°C.

[0021] Preferably, the preparation of the test solution comprises:

[0022] Step S101, sampling: accurately measure 1.0 mL of irinotecan hydrochloride injection sample using a pipette with an accuracy of ±0.05 mL and place it in a 10 mL brown volumetric flask;

[0023] Step S102, dilution: add 0.1% formic acid aqueous solution to the volumetric flask until it reaches the 8 mL mark, and vortex for 10 seconds to thoroughly mix the sample and the dilution solvent;

[0024] Step S103, constant volume: continue to add 0.1% formic acid aqueous solution to the 10 mL mark, and vortex again for 10 seconds to ensure a uniform solution;

[0025] Step S104, filtration: transfer the above solution to a syringe filter with a built-in 0.22 μm hydrophilic PTFE filter membrane, slowly push filter, and collect the filtrate as the test solution.

[0026] Preferably, the method further comprises preparing a reference solution:

[0027] (1) Preparation of stock solution:

[0028] Accurately weigh 10.00 mg of irinotecan hydrochloride reference substance and place it in a 10 mL brown volumetric flask. Add diluent solvent to dissolve and dilute to volume. Shake well to obtain stock solution a with a concentration of 1.00 mg / mL.

[0029] Accurately weigh 1.00 mg of SN-38 reference substance into a 10 mL brown volumetric flask. Dissolve it in diluent and dilute to volume. Shake well to obtain stock solution b with a concentration of 0.100 mg / mL.

[0030] Accurately weigh 0.50 mg of process impurity A reference substance and place it in a 10 mL brown volumetric flask. Add diluent solvent to dissolve and adjust to volume. Shake well to obtain stock solution C with a concentration of 0.050 mg / mL.

[0031] Accurately weigh 0.50 mg of degradation impurity B reference substance and place it in a 10 mL brown volumetric flask. Add diluent solvent to dissolve and adjust to volume. Shake well to obtain stock solution d with a concentration of 0.050 mg / mL.

[0032] (2) Preparation of mixed reference solution:

[0033] Accurately measure 10 μL, 20 μL, 50 μL, 100 μL, and 200 μL of each of the stock solutions a to d, respectively, and place them in a 10 mL brown volumetric flask. Add diluent to the volume and shake well to obtain mixed reference solutions of a series of concentrations.

[0034] Preferably, the method further comprises calculating the recovery rate:

[0035] Take irinotecan hydrochloride injection of known content, accurately measure 1.0 mL, add the mixed reference solution, and follow steps 101-104 to obtain the spiked test solution;

[0036] Inject sample for analysis and calculate the recovery rate of each impurity according to the formula: recovery rate = (measured amount - original amount in sample) / spiked amount × 100%;

[0037] The recovery rates of each impurity were between 95% and 105%, n=9, and RSD≤2.0%, where n is the number of repetitions, and RSD is used to measure the degree of dispersion of the recovery rate data of n times.

[0038] Preferably, the components detected include irinotecan hydrochloride and 7-ethyl-10-hydroxycamptothecin;

[0039] Preferably, the quantitative method is an external standard method, with a linear range of 0.05 μg / mL to 50 μg / mL and a correlation coefficient r≥0.999.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The combination of a core-shell column and a gradient elution procedure achieved complete separation of the main component, irinotecan hydrochloride, process impurities (such as A), degradation impurities (such as B), and SN-38. By optimizing chromatographic conditions, mobile phase system, and pretreatment process, comprehensive, rapid, and highly sensitive separation and detection of the main component, process impurities, degradation impurities, and related substances were achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 It is a schematic flow chart of the steps of the present invention. DETAILED DESCRIPTION

[0043] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0044] The following is an explanation of the method for detecting the components of irinotecan hydrochloride injection based on liquid chromatography according to the technical solution of the invention:

[0045] The sample pretreatment method is as follows:

[0046] Take a sample of irinotecan hydrochloride injection (specification: 2 mL: 40 mg), accurately measure 1.0 mL, place it into a 10 mL volumetric flask, dilute to the mark with 0.1% formic acid, shake well, filter through a 0.22 μm hydrophilic PTFE filter, and use the filtrate as the test solution. Separately, accurately weigh appropriate amounts of irinotecan hydrochloride reference substance, SN-38 reference substance, process impurity A (synthetic intermediate, 7-ethyl-10-[4-(1-piperidinyl)-1-butynyl]camptothecin) reference substance, and degradation impurity B (photolysis product, 10-(4-aminobutyl)-camptothecin) reference substance. Dissolve and dilute with 0.1% formic acid to prepare a series of mixed reference solutions (concentration range: 0.05 μg / mL to 50 μg / mL).

[0047] It should be noted that 0.1% formic acid aqueous solution can inhibit the dissociation of weakly acidic impurities (such as SN-38) and improve their retention on reversed-phase chromatography columns; hydrophilic PTFE filter membranes can prevent organic solvent residues from contaminating the chromatographic system.

[0048] The chromatographic conditions are as follows:

[0049] Use a core-shell chromatographic column (such as Waters ACQUITY UPLC BEH C18, 2.1 mm × 100 mm, 1.7 μm).

[0050] Mobile phase: 0.05% trifluoroacetic acid (TFA) aqueous solution as mobile phase A, acetonitrile as mobile phase B;

[0051] Gradient elution program:

[0052] 0-2 min, mobile phase B volume fraction 10%;

[0053] From 2 to 8 min, the volume fraction of mobile phase B was linearly increased to 30%;

[0054] From 8 to 15 min, the volume fraction of mobile phase B was linearly increased to 50%;

[0055] From 15 to 18 min, the volume fraction of mobile phase B increased linearly to 90%;

[0056] 18-20 min, the volume fraction of mobile phase B was maintained at 90%;

[0057] From 20 to 22 minutes, the volume fraction of mobile phase B was linearly reduced to 10%. Equilibrated to the initial state;

[0058] Flow rate: 0.35 mL / min;

[0059] Column temperature: 35°C;

[0060] Detection wavelength: 254nm (maximum absorption wavelength of the main component and most impurities) and 280nm (characteristic absorption wavelength of SN-38) dual wavelength switching detection.

[0061] It should be noted that: core-shell chromatographic columns use thin-shell fillers to reduce mass transfer resistance and improve separation efficiency (theoretical plate number can reach more than 10,000 / m); gradient elution can take into account the separation of strongly retained impurities (such as process impurity A) and weakly retained impurities (such as the main component); dual-wavelength detection can simultaneously meet the maximum absorption requirements of different impurities, thereby improving detection sensitivity.

[0062] System adaptability experiment:

[0063] Irinotecan hydrochloride reference solution (concentration: 10 μg / mL) was injected six times continuously, the chromatogram was recorded, and the theoretical plate number, tailing factor, and repeatability of the main component peak were calculated: theoretical plate number ≥ 8000 / m, tailing factor (T) ≤ 1.2, and peak area RSD ≤ 1.5%;

[0064] Inject the mixed reference solution, and the resolution (R) between the main component peak and the adjacent impurity peak (such as SN-38) should be ≥1.8.

[0065] Quantitative method: Use the external standard method to draw a standard curve with the peak area of ​​the reference solution as the ordinate and the concentration as the abscissa. Calculate the content of each component in the test solution. For trace impurities (such as a single impurity ≤0.1%), use the spiked recovery method to verify accuracy (recovery range: 95% to 105%).

[0066] Preparation of test solution:

[0067] Experimental materials and instruments

[0068] Sample: Irinotecan hydrochloride injection (specification: 2mL:40mg);

[0069] Dilution solvent: 0.1% formic acid aqueous solution (preparation method: take 0.1 mL of formic acid, add purified water to 1000 mL, shake well, and filter through a 0.22 μm microporous membrane for sterilization);

[0070] Filter membrane: 0.22 μm hydrophilic polytetrafluoroethylene (PTFE) filter membrane (material: polytetrafluoroethylene, pore size 0.22 μm);

[0071] Instruments: pipette (or precision pipette, range 1.0 mL), 10 mL volumetric flask (Grade A, brown, light-proof), vortex oscillator, filtration device (syringe filter).

[0072] The specific preparation steps are as follows:

[0073] Step S101, sampling: accurately measure 1.0 mL of irinotecan hydrochloride injection sample using a pipette with an accuracy of ±0.05 mL and place it in a 10 mL brown volumetric flask;

[0074] Step S102, dilution: add 0.1% formic acid aqueous solution to the volumetric flask until it reaches the 8 mL mark, and vortex for 10 seconds to thoroughly mix the sample and the dilution solvent;

[0075] Step S103, constant volume: continue to add 0.1% formic acid aqueous solution to the 10 mL mark, and vortex again for 10 seconds to ensure a uniform solution;

[0076] Step S104, filtration: transfer the above solution to a syringe filter with a built-in 0.22 μm hydrophilic PTFE filter membrane, slowly push filter, and collect the filtrate as the test solution.

[0077] It should be noted that the dilution solvent must be prepared immediately before use to avoid formic acid volatilization affecting the pH value (the pH should be controlled between 2.5 and 3.5 to ensure the inhibition of the dissociation of weakly acidic impurities such as SN-38). The volumetric flask must be pre-rinsed three times with the dilution solvent to avoid interference from dissolved substances. During filtration, the first 2 mL of initial filtrate (about 5 drops) must be discarded to ensure that impurities adsorbed on the filter membrane are fully washed to avoid affecting subsequent testing. The test solution must be sampled and analyzed within 30 minutes after preparation. If it cannot be tested in time, it must be refrigerated (4°C) for no more than 24 hours.

[0078] The preparation of reference solution is for adaptability testing and quantitative calibration.

[0079] Reference Material Information:

[0080] Main component reference substance: irinotecan hydrochloride (purity ≥99.5%).

[0081] Impurity reference substances: SN-38 (7-ethyl-10-hydroxycamptothecin, purity ≥98.0%); process impurity A (7-ethyl-10-[4-(1-piperidinyl)-1-butynyl]camptothecin, synthetic intermediate, purity ≥99.0%); degradation impurity B (10-(4-aminobutyl)-camptothecin, photolysis product, purity ≥97.5%).

[0082] Experimental materials and instruments: Dilution solvent: same as the test solution (0.1% formic acid aqueous solution); Instruments: analytical balance (precision 0.01 mg), 10 mL brown volumetric flask (Class A), pipette (range 100 μL~1 mL), vortex oscillator.

[0083] The specific steps are as follows:

[0084] (1) Preparation of stock solution:

[0085] Accurately weigh 10.00 mg of irinotecan hydrochloride reference substance and place it in a 10 mL brown volumetric flask. Add diluent solvent to dissolve and dilute to volume. Shake well to obtain stock solution a with a concentration of 1.00 mg / mL.

[0086] Accurately weigh 1.00 mg of SN-38 reference substance into a 10 mL brown volumetric flask. Dissolve it in diluent and dilute to volume. Shake well to obtain stock solution b with a concentration of 0.100 mg / mL.

[0087] Accurately weigh 0.50 mg of process impurity A reference substance and place it in a 10 mL brown volumetric flask. Add diluent solvent to dissolve and adjust to volume. Shake well to obtain stock solution C with a concentration of 0.050 mg / mL.

[0088] Accurately weigh 0.50 mg of degradation impurity B reference substance and place it in a 10 mL brown volumetric flask. Add diluent solvent to dissolve and adjust to volume. Shake well to obtain stock solution d with a concentration of 0.050 mg / mL.

[0089] (2) Preparation of mixed reference solution:

[0090] Accurately measure 10 μL, 20 μL, 50 μL, 100 μL, and 200 μL of each of the stock solutions a to d, respectively, and place them in a 10 mL brown volumetric flask. Add diluent to the volume and shake well to obtain mixed reference solutions of a series of concentrations.

[0091]

[0092] The concentration ranges of the impurities in the final mixed reference solution are: irinotecan hydrochloride (0.10-2.00 μg / mL), SN-38 (0.002-0.040 μg / mL), process impurity A (0.001-0.020 μg / mL), and degradation impurity B (0.001-0.020 μg / mL). The reference solution should be stored in a desiccator (below 25°C, humidity ≤ 60%) to avoid moisture absorption and decomposition. After preparation, the stock solution should be refrigerated (4°C) with a shelf life of no more than one month. The mixed reference solution should be prepared immediately prior to use to avoid degradation of impurities caused by prolonged storage. A calibrated analytical balance should be used for weighing, ensuring a weighing error of ≤±0.02 mg.

[0093] In conjunction with the recovery experiment, accurately measure 1.0 mL of irinotecan hydrochloride injection with a known content, add the mixed reference solution, and proceed according to steps 101-104 to obtain a spiked test solution. Sample injection analysis is performed, and the recovery of each impurity is calculated according to the formula: recovery = (measured amount - original amount in sample) / spiked amount × 100%; the recovery of each impurity is between 95% and 105%, n = 9, and the RSD is ≤ 2.0%, where n is the number of replicates. The RSD is used to measure the dispersion of the recovery data for n times.

[0094] The invention will be further described below based on specific embodiments:

[0095] Example 1

[0096] Chromatographic condition optimization: Verify that the core-shell column and gradient elution procedure improve the separation effect. The method is as follows:

[0097] A traditional C18 column (Agilent ZORBAX SB-C18, 4.6 mm × 250 mm, 5 μm) and a core-shell C18 column (Waters ACQUITY UPLC BEH C18, 2.1 mm × 100 mm, 1.7 μm) were used to inject the mixed reference solution under the same mobile phase (acetonitrile-0.05% TFA aqueous solution) and gradient program, and the separation of each component was compared.

[0098] The comparison of separation effects between traditional C18 column and core-shell C18 column is shown in Table 1:

[0099] Table 1

[0100] As shown in Table 1 above, the traditional C18 column has a resolution of 1.2 between SN-38 and the main component (not meeting the standard), the retention time of process impurity A is too long (25 minutes), and the total analysis time is 60 minutes. The core-shell C18 column has a resolution of 2.1 between SN-38 and the main component (meeting the standard), the retention time of process impurity A is 12 minutes, the total analysis time is 22 minutes, and the number of theoretical plates has increased threefold (from 2500 / m to 8000 / m).

[0101] Example 2

[0102] Dual-wavelength detection sensitivity verification. Compare the detection capabilities of single-wavelength (254nm) and dual-wavelength (254nm / 280nm) detection for SN-38. The method is as follows:

[0103] A series of SN-38 reference solutions (0.01 μg / mL to 10 μg / mL) were prepared. Detection was performed using a single wavelength of 254 nm and a dual wavelength of 254 nm / 280 nm. Peak areas were recorded, and the LOD and LOQ were calculated. The results are shown in Table 2.

[0104] Table 2

[0105] As shown in Table 2 above, for single wavelength (254nm): LOD = 0.2ng / mL, LOQ = 0.5ng / mL; for dual wavelength (254nm / 280nm): LOD = 0.05ng / mL, LOQ = 0.1ng / mL (sensitivity increased by 4 times).

[0106] Example 3

[0107] For actual sample testing, a batch of irinotecan hydrochloride injection was prepared with the test solution according to the above-mentioned "sample pretreatment method", sample injection and analysis were performed according to the "chromatographic conditions", the chromatogram was recorded, and the content of each component was calculated by the external standard method, as shown in Table 3 below.

[0108] Table 3

[0109] The above description provides a clear understanding of the specific embodiments of the present invention and the resulting technical benefits. The following describes the specific steps for the solid phase extraction (SPE) pretreatment for component detection in irinotecan hydrochloride injection according to the present invention, taking into account the physicochemical properties (e.g., polarity, pKa values) of the target components (irinotecan hydrochloride, SN-38, process impurities, and degradation impurities) and matrix interferences (e.g., buffer salts, surfactants, etc.), and details the operational procedures and key parameters.

[0110] The selection of SPE columns is based on the polarity of the target components and the matrix interference characteristics. A reversed-phase C18 silica bonded phase SPE column (such as Waters Oasis HLB or Agilent SPE-C18) is selected. The specific parameters are as follows:

[0111] Packing type: high purity silica bonded C18 (non-polar to medium polar retention);

[0112] Filler particle size: 40-60 μm (taking into account both retention efficiency and flow rate);

[0113] Column capacity: 100 mg / 6 mL (applicable to 1-5 mL sample volume);

[0114] Pretreatment requirements: Activate with methanol and water before use to ensure the filler is moist.

[0115] SPE column pretreatment steps:

[0116] Sample pretreatment, pH adjustment, and filtration: Irinotecan hydrochloride is a hydrochloride salt (pKa≈8.1), which is easily protonated (non-dissociated) under acidic conditions and is more easily retained by a reversed-phase C18 column. The buffer salts in the injection solution (such as sodium citrate, pH≈4.0-5.0) are highly polar interfering substances, and pH adjustment is required to further inhibit the dissociation of the target component and reduce matrix interference. The specific steps are as follows:

[0117] Take 1.0 mL of irinotecan hydrochloride injection sample (prepared in the same way as the test sample solution) and place it in a 10 mL plastic centrifuge tube;

[0118] Add 0.5 mL of 0.1 mol / L hydrochloric acid solution (adjust the pH to 2.5-3.0 to ensure that weakly acidic impurities such as irinotecan hydrochloride and SN-38 are fully protonated);

[0119] Vortex for 10 seconds and centrifuge at 10,000 rpm for 5 minutes (to remove possible precipitates or insoluble particles);

[0120] The supernatant was filtered through a 0.22 μm hydrophilic PTFE membrane (to prevent particles from clogging the SPE column), and the filtrate was collected for later use.

[0121] Activating the SPE column removes residual solvent and impurities, restoring the packing's retention capacity. Equilibration, on the other hand, allows the packing to reach a wet state suitable for retaining the target component. An SPE column (6 mL / 100 mg) was activated with 5 mL of methanol and then 5 mL of purified water (flow rate: 1-2 mL / min). After activation, the column was equilibrated with 5 mL of 0.1% formic acid in water (flow rate: 1-2 mL / min) to ensure that the polarity of the column environment was consistent with that of the sample solution.

[0122] Load the pretreated sample filtrate onto an SPE column, utilizing the differential partitioning between the stationary phase and mobile phase for separation of the target component and matrix interfering compounds. Specifically, place the activated and equilibrated SPE column onto a solid-phase extraction device with a 50 mL collection tube attached. Load the sample filtrate (1.0 mL) prepared in step 1 at a flow rate of 1-2 mL / min. Ensure the sample completely submerges the column packing during loading to avoid air bubbles.

[0123] Washing: Use a weakly or moderately polar solvent to remove matrix interferences (such as buffer salts, surfactants, and water-soluble impurities) that are not retained on the SPE column, while retaining the target component. Specifically, wash the SPE column with 5 mL of a 10% aqueous methanol solution (containing 0.1% formic acid) at a flow rate of 1-2 mL / min. Discard the wash solution (which contains most of the buffer salts, surfactants, and other interferences).

[0124] Elution: Use a highly polar organic solvent (such as a methanol-acetonitrile mixture) to elute the target components (irinotecan hydrochloride, SN-38, and impurities) retained on the SPE column. Collect the eluate for subsequent chromatographic analysis. Specifically, elute the SPE column with 5 mL of 80% acetonitrile in water (containing 0.1% formic acid) at a flow rate of 1-2 mL / min. Collect the eluate into a 10 mL brown volumetric flask (to ensure complete elution of the target components). Add 0.1% formic acid in water to the eluate, bring the volume to the mark, shake well, and filter through a 0.22 μm hydrophilic PTFE membrane. The filtrate is used as the test solution after SPE treatment.

[0125] It should be noted that if the SPE column needs to be reused, it can be rinsed with 5 mL of methanol and 5 mL of purified water in sequence, and then sealed and stored after drying.

[0126] Key parameter optimization:

[0127] Adjust the pH to 2.5-3.0. Irinotecan hydrochloride has a pKa of ≈8.1. When pH < 3, it mainly exists in a protonated form (non-dissociated state), with reduced polarity, making it more easily retained by a reversed-phase C18 column. Buffer salts (such as sodium citrate) have a reduced degree of dissociation under acidic conditions, increasing their water solubility and being effectively removed by the washing step.

[0128] The washing solvents were 10% methanol in water and 0.1% formic acid.

[0129] Methanol ratio (10%): weak polarity, can remove highly polar buffer salts (such as sodium citrate) and water-soluble surfactants;

[0130] 0.1% formic acid: maintains an acidic environment to prevent the target components from dissociating and losing during the washing process.

[0131] The elution solvent was 80% acetonitrile aqueous solution + 0.1% formic acid.

[0132] Acetonitrile ratio (80%): A highly polar organic solvent that can effectively elute retained neutral / weakly polar target components (such as irinotecan hydrochloride and SN-38);

[0133] 0.1% formic acid: inhibits the dissociation of target components and improves elution efficiency.

[0134] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting the components of irinotecan hydrochloride injection based on liquid chromatography, characterized in that: The following steps are involved: Step S100, sample pretreatment: taking irinotecan hydrochloride injection, diluting it with 0.1% formic acid aqueous solution, and filtering it through a 0.22 μm hydrophilic PTFE filter membrane to obtain a test solution; Step S200, chromatographic detection: using a core-shell C18 chromatographic column, with 0.05% trifluoroacetic acid aqueous solution as mobile phase A and acetonitrile as mobile phase B, performing gradient elution; Step S300, dual wavelength detection: simultaneous detection at wavelengths of 254 nm and 280 nm, and recording of chromatograms; Step S400, quantitative analysis: calculating the content of each component by external standard method.

2. The method for detecting the components of irinotecan hydrochloride injection based on liquid chromatography according to claim 1, characterized in that: The specifications of the core-shell C18 chromatographic column are: inner diameter 2.1 mm, column length 100 mm, and filler particle size 1.7 μm.

3. The method for detecting the components of irinotecan hydrochloride injection based on liquid chromatography according to claim 1, characterized in that: The gradient elution procedure is: 0-2 min, mobile phase B volume fraction 10%; From 2 to 8 min, the volume fraction of mobile phase B was linearly increased to 30%; From 8 to 15 min, the volume fraction of mobile phase B was linearly increased to 50%; From 15 to 18 min, the volume fraction of mobile phase B increased linearly to 90%; 18-20 min, the volume fraction of mobile phase B was maintained at 90%; From 20 to 22 min, the volume fraction of mobile phase B decreased linearly to 10%.

4. The method for detecting the components of irinotecan hydrochloride injection based on liquid chromatography according to claim 1, characterized in that: The column flow rate was 0.35 mL / min and the column temperature was 35 °C.

5. The method for detecting the components of irinotecan hydrochloride injection based on liquid chromatography according to claim 1, characterized in that: The preparation of the test solution comprises: Step S101, sampling: accurately measure 1.0 mL of irinotecan hydrochloride injection sample using a pipette with an accuracy of ±0.05 mL and place it in a 10 mL brown volumetric flask; Step S102, dilution: add 0.1% formic acid aqueous solution to the volumetric flask until it reaches the 8 mL mark, and vortex for 10 seconds to thoroughly mix the sample and the dilution solvent; Step S103, constant volume: continue to add 0.1% formic acid aqueous solution to the 10 mL mark, and vortex again for 10 seconds to ensure a uniform solution; Step S104, filtration: transfer the above solution to a syringe filter with a built-in 0.22 μm hydrophilic PTFE filter membrane, slowly push filter, and collect the filtrate as the test solution.

6. The method for detecting the components of irinotecan hydrochloride injection based on liquid chromatography according to claim 4, characterized in that: It also includes the preparation of reference solution: (1) Preparation of stock solution: Accurately weigh 10.00 mg of irinotecan hydrochloride reference substance and place it in a 10 mL brown volumetric flask. Add diluent solvent to dissolve and dilute to volume. Shake well to obtain stock solution a with a concentration of 1.00 mg / mL. Accurately weigh 1.00 mg of SN-38 reference substance and place it in a 10 mL brown volumetric flask. Dissolve it in diluent and adjust to volume. Shake well to obtain stock solution b with a concentration of 0.100 mg / mL. Accurately weigh 0.50 mg of process impurity A reference substance and place it in a 10 mL brown volumetric flask. Add diluent solvent to dissolve and adjust to volume. Shake well to obtain stock solution C with a concentration of 0.050 mg / mL. Accurately weigh 0.50 mg of degradation impurity B reference substance and place it in a 10 mL brown volumetric flask. Add diluent solvent to dissolve and adjust to volume. Shake well to obtain stock solution d with a concentration of 0.050 mg / mL. (2) Preparation of mixed reference solution: Accurately measure 10 μL, 20 μL, 50 μL, 100 μL, and 200 μL of each of the stock solutions a to d, respectively, and place them in a 10 mL brown volumetric flask. Add diluent to the volume and shake well to obtain mixed reference solutions of a series of concentrations.

7. The method for detecting the components of irinotecan hydrochloride injection based on liquid chromatography according to claim 5, characterized in that: Also includes recovery calculations: Take irinotecan hydrochloride injection of known content, accurately measure 1.0 mL, add the mixed reference solution, and follow steps 101-104 to obtain the spiked test solution; Inject sample for analysis and calculate the recovery rate of each impurity according to the formula: recovery rate = (measured amount - original amount in sample) / spiked amount × 100%; The recovery rate of each impurity is between 95% and 105%, n=9, RSD≤2.0%, where n is the number of repetitions, and RSD is used to measure the degree of dispersion of the recovery rate data of n times.

8. The method for detecting components of irinotecan hydrochloride injection based on liquid chromatography according to claim 1, characterized in that: The components of the test include irinotecan hydrochloride and 7-ethyl-10-hydroxycamptothecin.

9. The method for detecting components of irinotecan hydrochloride injection based on liquid chromatography according to claim 1, characterized in that: The quantitative method is an external standard method, the linear range is 0.05 μg / mL to 50 μg / mL, and the correlation coefficient r is ≥ 0.999.

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